Corneal infection is a sight-threatening disease of the eye and Pseudomonas aeruginosa remains a leading cause. The critical early steps in disease initiation include bacterial invasion of, and intracellular survival within, corneal epithelial cells. Processes involved in these steps are potential therapeutic targets for intervention in this disease. Research in this lab has found that the Type III Secretion System (T3SS) of P. aeruginosa is required for intracellular replication of P. aeruginosa within corneal epithelial cells involving escape from perinuclear vacuoles and trafficking to infection-induced bleb-niches. The focus of this proposal is on the pathogenesis of these T3SS-induced bleb-niches. The T3SS is known to inject effector proteins directly from the bacterial cytoplasm into the host cell. ExoY is the least studied T3SS effector of P. aeruginosa, but it is known to cause disruption of host cytoskeleton and cell rounding in vitro. The only known biochemical function of ExoY is its adenylate cyclase activity that promotes the accumulation of cyclic AMP in mammalian cells. The role of ExoY in corneal disease caused by P. aeruginosa has not been explored. The hypothesis to be tested is that adenylate cyclase activity of ExoY is required for ExoY-mediated P. aeruginosa escape from perinuclear vacuoles into infection-induced bleb-niches, and that this contributes to corneal disease in vivo. Supporting the hypothesis is preliminary data showing that wild-type ExoY, but not a mutant form of ExoY without adenylate cyclase activity, enables escape from perinuclear vacuoles. Aim 1 is to confirm the role of adenylate cyclase activity of ExoY in bleb-niche formation in cultured corneal epithelial cells. The approach will be to use wild- type ExoY and its mutant forms without adenylate cyclase activity, and chemical modulators of adenylate cyclase activity and of cyclic AMP levels, to examine their effects on bleb-niche formation in vitro. Aim 2 will examine the role of ExoY and its adenylate cyclase activity in bacterial escape from perinuclear vacuoles and in corneal disease in vivo. The approach will be to study the impact of ExoY mutations on intracellular localization within infected corneas using confocal microscopy. Corneal disease severity will be graded using an established scoring system and bacterial colonization quantified by viable counts of homogenized infected corneas.